Tuberculosis (TB) is the most common cause of morbidity/mortality in HIV-infected individuals in India, which has among the world's highest burden of HIV and TB. Since children acquire HIV/TB from their caregivers, active TB is highly prevalent (14-67.5%) among HIV-infected young children in India - a vulnerable population, where lack of early detection / prevention of TB often have fatal consequences. Diagnosis of TB is particularly challenging in young children. Due to similar clinical presentation, active TB cannot be reliably distinguished from HIV (or co-infections). Tuberculin skin test (TST) is often unreliable and bacterial confirmation (culture), when available, is difficult and does not provide a diagnosis for weeks to months. IFN-?-release assays (IGRA) measure T-cells in response to two antigens, ESAT-6 and CFP-10, which are strong targets of T-helper cells, and are encoded by Mycobacterium tuberculosis but not by BCG or most nontuberculous mycobacteria (NTB). IGRAs such as ELISpot are significantly more sensitive/specific than TST for the diagnosis of active TB in young children with high rates of HIV, malnutrition, BCG vaccination and NTB. They are also especially useful for diagnosing extra-pulmonary TB, which is prevalent in young children but often difficult to diagnose, particularly because specimen collection and yield is limited. We will build on a strong, existing Indo-US collaboration with Byramjee Jeejeebhoy Medical College (BJMC) and National AIDS Research Institute (NARI), both of which are NIH-approved HIV Clinical Trial Units (CTUs), focused on pediatric studies in Pune, India. Inpatient/outpatient clinics and laboratory facilities at these centers serve children with high burden of HIV/TB. Our collaborator Professor Ajit Lalvani (Imperial College, London) invented the ELISpot assay for TB. His laboratory has recently developed novel ELISpot assays that measure IFN-? release to TB antigens in addition to ESAT-6/CFP-10. Recent data from adults show that one such assay, ELISpotPLUS is highly promising, but there is no information on its applicability to childhood TB, particularly in context of HIV. In this study, we will evaluate an enhanced ELISpot (measures IFN-? release to several TB antigens in addition to ESAT-6/CFP-10), for the diagnosis of active TB in young children. Our existing Indo-US collaborations significantly defray study costs and provide "added value", which allow us to conduct this relatively large study, in a vulnerable population, with limited resources available through this R03 funding mechanism. PUBLIC HEALTH RELEVANCE: This study specifically addresses improved detection of TB in young children, a vulnerable and understudied population in India. We propose a highly collaborative Indo-US study that takes advantage of our clinical and laboratory strengths to investigate the utility and incremental diagnostic value of a novel assay for pediatric TB disease, a serious, burdensome and yet difficult to diagnose illness in young children. This study will result in transfer of technology and training for a novel TB diagnostics to Pune, India and would lead to implementation of early and effective TB control measures, limiting HIV disease progression and higher morbidity/mortality associated with TB co-infection.